Our long-term objective is to understand the signaling mechanisms and functions of heterotrimeric G proteins and G protein-coupled receptors. Recently we have discovered that G protein Gas can directly stimulate the activity of Src-family tyrosine kinases, and that tyrosine kinase Csk plays a critical role in linking signals from G proteins and G protein-coupled receptors to actin cytoskeletal reorganization. Yet, the relative contribution of Src-family tyrosine kinases and adenylyl cyclases to the physiology of Gs in animals is not clear. The mechanism by which Csk relays heterotrimeric G protein signals to Rho GTPases is not defined. In this grant application, we have proposed experiments to answer these questions. The specific aims are: 1. Cellular physiological studies of Gas regulation of Src. We focus on the role of the Gs-Src link in Gs-coupled f32-admergic receptor desensitization! internalization in cells. 2. Mouse genetic study of the physiological role of Gs-Src signaling. We will use two approaches to examine Gs-Src function in mouse. One is to generate knock-in mice with different Gas mutants. Another is to rescue the Gas-knock-out mice with different Gas mutants to investigate the rescue of specific phenotypes of the Gas knockout mice. 3. Signaling mechanism from G proteins to actin cytoskeletal reorganization. We will test a specific hypothesis that, downstream of Csk, Src and p190 RhoGAP are signal transducers leading to Rho in this G protein pathway. We will extend the cytoskeletal reorganization study in fibroblast cells to clinically relevant breast cancer cell migration. This research is directly related to human health. Mutations of G proteins, G protein-coupled receptors, and tyrosine kinases can lead to many human diseases such as cancers and heart diseases.